PAK4, a novel gene encoding a serine/threonine kinase

ABSTRACT

This invention provides an isolated mammalian nucleic acid molecule encoding a PAK4 serine/threonine kinase. This invention provides an isolated nucleic acid molecule encoding a mutant homolog of the mammalian PAK4 serine/threonine kinase whose amino acid sequence is set forth in FIG.  1 A (SEQ ID NO: 2). This invention provides a fusion protein comprising a PAK4 serine/threonine kinase or a fragment thereof and a second peptide. This invention provides a purified mammalian PAK4 serine/threonine kinase. This invention provides a protein comprising substantially the amino acid sequence set forth in FIG.  1 A. This invention provides a monoclonal antibody directed to an epitope of a PAK4 serine/threonine kinase. This invention provides a method of inhibiting PAK4 function comprising administering a ligand comprising an amino acid domain which binds to a GTP binding protein so as to inhibit binding of the GTP binding protein to PAK4. This invention provides a method of inhibiting PAK4 function comprising administering a ligand which binds to the GTP binding domain of PAK4 so as to inhibit PAK4 binding to a GTP binding protein. This invention provides a method of inhibiting PAK4 serine/threonine kinase function comprising administering a ligand which blocks an ATP binding domain so as to inhibit PAK4 serine/threonine kinase function. This invention provides a method of inhibiting growth of a tumor cell comprising blocking Cdc42Hs by administering a ligand capable of binding to a Cdc42Hs binding site of a PAK4 serine/threonine kinase.

This application is a continuation of PCT International Application No.PCT/US99/11341, filed May 21, 1999, designating the United States ofAmerica, which is a continuation-in-part and claims priority of U.S.Ser. No. 09/082,737, filed May 21, 1998 now U.S. Pat. No. 6,013,500issued Jan. 11, 2002, the contents of which are hereby incorporated byreference into the present application.

Throughout this application, various references are referred to withinparentheses. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citation for these references may be found at the end ofthis application, preceding the claims.

BACKGROUND OF THE INVENTION

The GTPases Rac and Cdc42Hs control diverse cellular functions. Inaddition to being mediators of intracellular signaling cascades, theyhave important roles in cell morphogenesis and mitogenesis. A novel PAKrelated kinase, PAK4, has been identified as a new effector molecule forCdc42Hs. PAK4 interacts only with the activated form of Cdc42Hs throughits GTPase binding domain (GBD). Co-expression of PAK4 and theconstitutively active Cdc42HsV12 cause the re-distribution of PAK4 tothe Brefeldin A sensitive compartment of the Golgi membrane and thesubsequent induction of filopodia and actin polymerization. Importantly,the reorganization of the actin cytoskeleton was dependent on PAK4kinase activity and on its interaction with Cdc42Hs. Thus, unlike othermembers of the PAK family, PAK4 provides a novel link between Cdc42Hsand the actin cytoskeleton. The cellular locations of PAK4 and Cdc42Hssuggests a role for the Golgi in cell morphogenesis.

Members of the Rho family of small GTPases Rac and Cdc42Hs have beenimplicated in diverse biological processes. These include roles in cellproliferation, progression through the cell cycle, and oncogenictransformation (Van Aelst and D'Souza-Schorey, 1997). Cdc42Hs and Racalso play important roles in signal transduction cascades such as thosethat lead to activation of both the JNK and the p38 families of MAPkinases, and thus lead to long term changes in gene expression (Bagrodiaet al., 1995; Brown et al., 1996; Coso et al., 1995; Minden et al.,1995; Zhang et al., 1995). One of the most important functions of Racand Cdc42Hs is the regulation of the organization of the actincytoskeleton. Microinjection of Cdc42Hs into fibroblasts and a varietyof other cell types causes the induction of filopodia protrusionsfollowed by the formation of lamellipodia. While the induction offilopodia is caused by Cdc42Hs activation, the induction of lamellipodiais probably due to the ability of Cdc42Hs to activate Rac. Thus,co-expression of Cdc42Hs with a dominant negative Rac mutant results inthe sustained induction of filopodia without the subsequent induction oflamellipodia. Furthermore, microinjection of activated Rac leads to theinduction of lamellipodia, but not filopodia. In addition to theformation of polymerized actin structures, both Cdc42Hs and Rac inducethe formation of focal complexes that are associated with the filopodiaand lamellipodia. Finally, in some cells, both Cdc42Hs and Rac have beenshown to have a role in the dissolution of stress fibers, which may bedue to antagonism between these GTPases and a third GTPase, RhoA.

A great deal of effort has been made to identify the downstreammolecular targets for Rac and Cdc42Hs. Several proteins were shown tointeract with the activated forms of Rac and Cdc42Hs including PAK65,p67-phox, WASP, IQGAP, and MLK3 (Manser et al. 1994, Martin et al. 1995;Bagrodia et al.; 1995, Aspenstrom et al., 1996; Rana et al., 1996;Symons et al., 1996; Hart et al. 1996; Kuroda et al. 1996; Teramoto etal., 1996; Burbelo et al. 1995; Van Aelst et al., 1996). PAK was thefirst protein kinase that was shown to be a target for Rac and Cdc42Hs,and consequently drew much attention. Activated Rac and Cdc42Hsstimulate PAK autophosphorylation and stimulate its kinase activity.Several PAK family members have been identified and all were shown tointeract with GTP bound forms of Rac and Cdc42Hs. These include humanPAK1 and 2, mouse PAK3, and the rat homologues PAK α, β, and γ (Brown etal., 1996; Manser et al., 1994; Martin et al., 1995; Bagrodia et al.;1995). The PAKs are all similar in structure, containing an aminoterminal regulatory domain and a carboxyl terminal kinase domain. Theyare also all quite similar in sequence, exhibiting 73% overall sequenceidentity and approximately 92% sequence identity within the kinasedomain (Sells and Chernof, 1997). The regulatory domains of the PAKscontain a GTPase binding domain GBD (Symons et al., 1996) (known also asCdc42Hs/Rac Interactive Binding (CRIB) domain) (Burbelo et al., 1995)that is necessary and essential for their direct interaction with bothCdc42Hs and Rac.

The functions of the PAKs are not yet entirely known. The sequencesimilarities between the PAKs and yeast STE20, however, suggests a rolein transcription activation or cell morphogenesis. In Saccharomycescerevisiae, STE20 is activated by Cdc42p, and is an important componentof the KSS/FUS3 MAP Kinase pathway. STE20 and the related CLA4 may alsomediate cytoskeletal changes induced by Cdc42p, such as those that occurduring cytokinesis. Because of the evolutionary conservation betweenmany yeast and mammalian signaling enzymes, it seems likely that thePAKs may have functions similar to the yeast STE20 and CLA4 proteins. Infact, the PAKs have been shown to weakly activate the JNK MAP kinasepathway in some cells (Bagrodia et al., 1995; Brown et al., 1996; Zhanget al., 1995). This suggests that, like STE20, the PAKs may be involvedin MAP Kinase pathways. Some groups have shown however, that the PAKsare not necessary for JNK activation, and thus their roles in MAP Kinasepathways are as yet unclear. The PAKs may also be involved incytoskeletal organization. PAK1 was reported to induce filopodia andmembrane ruffles similar to those induced by Cdc42Hs and Rac, and tolocalize to polymerized actin. Interestingly however, these cytoskeletalchanges are partly independent of PAK1's kinase activity, and they alsooccur independently of PAK1's ability to bind the Rho GTPases. Thus,while overexpressed PAK1 can promote cytoskeletal changes, it may notspecifically mediate the cytoskeletal changes induced by Rac andCdc42Hs. Others have found that PAK1 does not induce filopodia orlamellipodia but instead has a role in the dissolution of stress fibersand down-regulation of focal adhesions. Finally, effector mutants of Racand Cdc42Hs, (such as RacL61(Y40C) and Cdc42HsL61(Y40C)) that do notbind the PAKs, maintain the ability to induce lamellopodia andfilopodia. Taken together, these results suggest that the induction oflamellipodia and filopodia by Rac and Cdc42Hs can occur independently ofthe known PAKs.

Here the cloning and characterization of a novel serine/threoninekinase, PAK4 is reported. Like other members of the PAK family, PAK4contains an amino terminal regulatory domain and a carboxyl terminalkinase domain. The kinase domain of PAK4 shares 53% sequence identitywith those of the other PAKs. Outside of this region however, PAK4 isentirely different in sequence from the other PAKs, except for a shortstretch containing a modified GBD motif. PAK4 is the first member of thePAK family to be identified that differs significantly in sequence fromthe other PAKs, and thus represents an entirely new member of the PAKfamily. PAK4 interacts specifically with the GTP bound form of Cdc42Hsvia its GBD motif and weakly activates the JNK family of MAP kinases.Co-expression of PAK4 with Cdc42Hs causes PAK4 to translocate from adiffuse perinuclear area to the Golgi membrane and subsequently inducedthe formation of filopodia and actin polymerization. Thus, the Golgitranslocation of PAK4 by Cdc42Hs may be important for its ability toinduce filopodia. Furthermore, PAK4 interacts with the Cdc42Hs effectormutant, Cdc42HsL61(Y40C) that was previously was shown to inducefilopodia independently of PAKs. These results indicate therefore, thatPAK4, rather than the previously identified PAKs, provides a linkbetween Cdc42Hs and the actin cytoskeleton.

SUMMARY OF THE INVENTION

This invention provides an isolated mammalian nucleic acid moleculeencoding a PAK4 serine/threonine kinase.

This invention provides an isolated nucleic acid molecule encoding amutant homolog of the mammalian PAK4 serine/threonine kinase whose aminoacid sequence is set forth in FIG. 1A (SEQ ID NO: 2).

This invention provides a fusion protein comprising a PAK4serine/threonine kinase or a fragment thereof and a second peptide.

This invention provides a purified mammalian PAK4 serine/threoninekinase.

This invention provides a protein comprising substantially the aminoacid sequence set forth in FIG. 1A.

This invention provides a monoclonal antibody directed to an epitope ofa PAK4 serine/threonine kinase.

This invention provides a method of inhibiting PAK4 function comprisingadministering a ligand comprising an amino acid domain which binds to aGTP binding protein so as to inhibit binding of the GTP binding proteinto PAK4.

This invention provides a method of inhibiting PAK4 function comprisingadministering a ligand which binds to the GTP binding domain of PAK4 soas to inhibit PAK4 binding to a GTP binding protein.

This invention provides a method of inhibiting PAK4 serine/threoninekinase function comprising administering a ligand which blocks an ATPbinding domain so as to inhibit PAK4 serine/threonine kinase function.

This invention provides a method of inhibiting growth of a tumor cellcomprising blocking Cdc42Hs by administering a ligand capable of bindingto a Cdc42Hs binding site of a PAK4 serine/threonine kinase.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E. Sequence and expression pattern of PAK4.

FIGS. 1A-1B Nucleotide sequence of PAK4, a novel serine/threonine kinase(SEQ ID NO: 1 and SEQ OD NO: 2). The CRIB domain (amino acids 1-30) andthe kinase domain (amino acids 323-574) are underlined. FIG. 1C.Alignment of the kinase domain of PAK4 with those of PAK2 and STE20 (SEQID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively). FIG. 1D.Alignment of the GBD/CRIB motif of PAK4 (SEQ ID NO: 6) with thecorresponding regions of several other mammalian (PAK2 (SEQ ID NO: 7)and WASP (SEQ ID NO: 9)) and yeast (STE20 (SEQ ID NO: 8) and CLA4 (SEQID NO: 10)) Rac and Cdc42Hs interacting proteins. FIG. 1E. Northern blotanalysis of PAK4. A Northern blot containing mRNA from various humantissues was probed with a cDNA containing the kinase domain of PAK4. Aband of approximately 3 KB is indicated.

FIGS. 2A-2B. PAK4 interacts with the activated Cdc42Hs through itsGBD/CRIB domain.

FIG. 2A. Recombinant PAK4 and hPAK2 (2-3 μg) were analyzed by theoverlay assay and probed with the indicated GTPase preloaded with either[γ³²P]GTP or ³²[βP]GDP as described in materials and methods. FIG. 2B.Cos-1 cells were transiently transfected with expression vectorscontaining HA tagged PAK4 or PAK4ΔGBD, (a deletion mutant lacking theGBD/CRIB domain). After transient expression, Cos-1 cells wereharvested, immunopurified with the anti HA antibodies, separated by SDSPAGE and transferred to nylon membranes. Binding of PAK4 or PAK4DGBD to[γ³²P]GTP loaded Cdc42Hs was assessed as in FIG. 2A. To ensure that bothwild-type PAK4 and PAK4AGBD were expressed at approximately equivalentlevels, cell extract (25 μg) was analyzed by Western Blots probed withanti HA antibody.

FIGS. 3A-3B. Analysis of PAK4 kinase activity.

FIG. 3A. NIH3T3 cells were transfected with either empty Srα vector(control) or with increasing doses of Srα expression vectors containingthe PAK cDNA fused to an HA epitope tag (0.5 and 1 μg). After transientexpression, PAK4 was immunopurified from cell lysates using anti HAantibody. Immunopurified PAK4 was incubated with Histone H4, or withoutany substrate in the presence of [γ-³²P]ATP and kinase buffer (Minden etal., 1994). Substrate phosphorylation was analyzed after SDS PAGE andautoradiography. Autophosphorylation of the 70 kD PAK4 and a band ofapproximately 43 kDa that co-purifies with PAK4 is indicated. FIG. 3B.Cos-1 cells were transfected with 1 μg of expression vectors containingeither wild-type PAK4 or the indicated PAK4 mutants, all fused to HAtags. After transient expression, immunopurified PAK4 was used tophosphorylate Histone H4 in the presence of kinase buffer and[γ-³²P]ATP. Substrate phosphorylation is indicated. Similar results wereobtained with NIH3T3 cells. FIGS. 3C-3E. NIH3T3 cells were transfectedwith empty Srα vector (control) or increasing doses of Srα myc taggedPAK4 expression vector (0.2, 0.6, or 1 μg) together with 1 μg of HAtagged JNK, ERK, or p38 expression vectors. As positive controls, cellswere either transfected with expression vectors for Rac2L61 or RasV12 ortreated with 400 mM sorbitol, as indicated. After transient expression,JNK, ERK, or p38 were immunopurified from cell lysates using anti HAantibody. Immunopurified proteins were incubated in kinase buffer and[γ-³²P]ATP together with either GST-cJun, GST-ATF2, or MBP assubstrates, respectively (Minden et al., 1995). Substratephosphorylation was visualized after SDS PAGE and autoradiography.Substrate phosphorylation was quantitated by phosphorimager analysis.The numbers indicated are the averages of three independent experiments.

FIGS. 4A-4J. Localization of PAK4 and its effects on the actincytoskeleton.

FIGS. 4A-4F. Porcine endothelial cells (PAE) were microinjected with HAtagged PAK4 expression vector alone or in combination with Myc taggedCdc42HsV12 or RacV12 expression vectors (50 ng/μl plasmid). Cell werefixed 11-14 hours after injection and PAK4 was visualized byimmunofluorescence microscopy after staining with FITC tagged HAantibody. Polymerized actin was visualized after staining with Rhodamineconjugated phalloidin. FIGS. 4G-4J. PAE cells were microinjected withMyc tagged PAK2 expression vector alone and in combination withCdc42HsV12 or RacV12 expression vectors. Polymerized actin wasvisualized as described in FIGS. 4A-4F. Arrows indicate the injectedcells.

FIGS. 5A-5H PAK4 is recruited by Cdc42Hs to the BFA sensitivecompartment of the Golgi.

PAE cells were microinjected with HA tagged PAK4 expression vector aloneor in combination with Cdc42HsV12 expression vector. After 12-16 hourscells were either left untreated or treated with 5 μg/ml BFA for 3-5min. at 37° C. followed immediately by fixation. b-COP was visualized bya specific antibody against b-COP protein (Gift of Richard Khan). Tocompare the localization of PAK4 to b-COP compare fluorescencemicrograph of anti HA staining to that of anti b-COP staining.

FIGS. 6A-6H. PAK4 localization to the Golgi and kinase activity areessential for actin polymerization.

Fluorescence micrographs of PAE cells microinjected with expressionvectors containing HA tagged kinase inactive PAK4(M430) or thePAK4(E474) mutant, alone or in combination with Cdc42HsV12 expressionvector. PAK4 was detected by HA antibody and polymerized actin wasvisualized by phalloidin staining.

FIGS. 7A-7C. Analysis of Cdc42Hs effector mutants.

FIG. 7A. Recombinant PAK4, hPAK2 , and WASP (2-3 μg) were analyzed bythe overlay assay and probed with the indicated Cdc42Hs mutant preloadedwith [γ³²P]GTP (Martin et al., 1995). FIGS. 7B-7C. Porcine endothelialcells (PAE) were injected with HA tagged PAK4 expression vector alone orin combination with Myc tagged Cdc42HsL61(C40) mutant. (100 ng/μlplasmid). Cell were fixed 11-14 hours after injection and PAK4 wasvisualized by immunofluorescence microscopy after staining with FITCtagged HA antibody.

FIGS. 8A-8C FIG. 8A. Partial cDNA of the mouse PAK4 (SEQ ID NO: 13).FIG. 8B. Predicted amino acid sequence of the partial mouse PAK4 cDNA(SEQ ID NO: 14). FIG. 8C. Partial genomic sequence of mouse PAK4 (SEQ IDNO: 15).

DETAILED DESCRIPTION OF THE INVENTION

The following standard abbreviations are used throughout thespecification to indicate specific nucleotides:

C=cytosine A=adenosine

T=thymidine G=guanosine

This invention provides an isolated nucleic acid molecule encoding aPAK4 serine/threonine kinase. In an embodiment the isolated nucleic acidmolecule encoding a PAK4 serine/threonine kinase is a DNA molecule. Inanother embodiment the isolated nucleic acid molecule encoding a PAK4serine/threonine kinase is a cDNA molecule. In a further embodiment theisolated DNA molecule encoding a PAK4 serine/threonine kinase is agenomic DNA molecule. In an embodiment the isolated nucleic acidmolecule encoding a PAK4 serine/threonine kinase is an RNA molecule.

The DNA molecules of the subject invention also include DNA moleculescoding for polypeptide analogs, fragments or derivatives of antigenicpolypeptides which differ from naturally-occurring forms in terms of theidentity or location of one or more amino acid residues (deletionanalogs containing less than all of the residues specified for theprotein, substitution analogs wherein one or more residues specified arereplaced by other residues and addition analogs where in one or moreamino acid residues is added to a terminal or medial portion of thepolypeptides) and which share some or all properties ofnaturally-occurring forms. These molecules include: the incorporation ofcodons “preferred” for expression by selected non-mammalian hosts; theprovision of sites for cleavage by restriction endonuclease enzymes; andthe provision of additional initial, terminal or intermediate DNAsequences that facilitate construction of readily expressed vectors.

The DNA molecules described and claimed herein are useful for theinformation which they provide concerning the amino acid sequence of thepolypeptide, PAK4 serine/threonine kinase, and as products for the largescale synthesis of the polypeptide (PAK4 serine/threonine kinase) by avariety of recombinant techniques. The molecule is useful for generatingnew cloning and expression vectors, transformed and transfectedprokaryotic and eukaryotic host cells, and new and useful methods forcultured growth of such host cells capable of expression of thepolypeptide (PAK4 serine/threonine kinase) and related products.

In another embodiment of the isolated nucleic acid molecule encoding aPAK4 serine/threonine kinase, the nucleic acid molecule encodes amammalian PAK4 serine/threonine kinase. In a preferred embodiment of theisolated nucleic acid molecule encoding a PAK4 serine/threonine kinase,the mammalian PAK4 serine/threonine kinase is a human, mouse or rat PAK4serine/threonine kinase. In a further embodiment of the isolated nucleicacid molecule encoding a mammalian PAK4 serine/threonine kinase, thenucleic acid molecule encodes a PAK4 serine/threonine kinase comprisingan amino acid sequence as set forth in FIG. 1A (SEQ ID NO: 2). In anembodiment of the isolated nucleic acid molecule encoding a PAK4serine/threonine kinase comprising an amino acid sequence as set forthin FIG. 1A (SEQ ID NO: 2), the encoded amino acid sequence comprises aGTPase binding domain (GBD). In another embodiment of the isolatednucleic acid molecule encoding the mammalian PAK4 serine/threoninekinase, the PAK4 serine/threonine kinase has substantially the sameamino acid sequence as set forth in FIG. 1A (SEQ ID NO: 2).

In a further embodiment of the isolated nucleic acid molecule encodingthe mammalian PAK4 serine/threonine kinase, the encoded PAK4serine/threonine kinase has the amino acid sequence as set forth in FIG.1A (SEQ ID NO: 2).

This invention provides an isolated nucleic acid molecule encoding amutant homolog of the mammalian PAK4 serine/threonine kinase whose aminoacid sequence is set forth in FIG. 1A (SEQ ID NO: 2). In an embodimentthe isolated nucleic acid molecule encoding the above-described mutanthomolog of the mammalian PAK4 serine/threonine kinase is a deletionmutant. In a further embodiment of the deletion mutant, the encodedmutant homolog comprises a GTPase binding domain. In a still furtherembodiment of the deletion mutant, the encoded mutant homolog does notcomprise a GTPase binding domain. In an embodiment of the isolatednucleic acid molecule encoding the mammalian PAK4 serine/threoninekinase, the mammalian PAK4 serine/threonine kinase comprises the nucleicacid sequence set forth in FIG. 1A (SEQ ID NO:1).

This invention provides a fusion protein comprising a PAK4serine/threonine kinase or a fragment thereof and a second peptide.Fusion of a peptide with a smaller peptide, for example, a tag such as ahemaglutinin (HA) or myc tag, is well known to one of ordinary skill inthe art.

This invention provides a vector comprising the mammalian nucleic acidmolecule encoding a PAK4 serine/threonine kinase. In an embodiment thevector is adapted for expression in a host cell which comprises theregulatory elements necessary for expression of the nucleic acidmolecule in the host cell operatively linked to the nucleic acidmolecule encoding the PAK4 serine/threonine kinase as to permitexpression of the PAK4 serine/threonine kinase. In another embodiment ofthe vector, the host cell is a eukaryotic, bacterial, insect or yeastcell. In a further embodiment of the vector, the eukaryotic host cell isa mammalian cell. In a still further embodiment the vector is a plasmid.

This invention also provides a vector comprising the nucleic acidmolecule encoding a mammalian PAK4 serine/threonine kinase, wherein thenucleic acid molecule is a cDNA molecule. In an embodiment the vector isadapted for expression in a host cell which comprises the regulatoryelements necessary for expression of the nucleic acid molecule in thehost cell operatively linked to the nucleic acid molecule encoding thePAK4 serine/threonine kinase as to permit expression of the PAK4serine/threonine kinase. In another embodiment of the vector, the hostcell is a eukaryotic, bacterial, insect or yeast cell. In a furtherembodiment of the vector, the eukaryotic host cell is a mammalian cell.In a still further embodiment the vector is a plasmid.

In an embodiment, a full-length cDNA nucleic acid molecule encoding ahuman PAK4 serine/threonine kinase is inserted by subcloning into a SRαplasmid and the resulting plasmid is designated as SRαHAPAK4. PlasmidSRαHAPAK4 was deposited on May 21, 1998 with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209,U.S.A. under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. Plasmid SrαHAPAK4 was accorded ATCC Accession Number 209888.

Numerous vectors for expressing the inventive proteins may be employed.Such vectors, including plasmid vectors, cosmid vectors, bacteriophagevectors and other viruses, are well known in the art. For example, oneclass of vectors utilizes DNA elements which are derived from animalviruses such as bovine papilloma virus, polyoma virus, adenovirus,vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MoMLV), SemlikiForest virus or SV40 virus. Additionally, cells which have stablyintegrated the DNA into their chromosomes may be selected by introducingone or more markers which allow for the selection of transfected hostcells. The markers may provide, for example, prototrophy to anauxotrophic host, biocide resistance or resistance to heavy metals suchas copper. The selectable marker gene can be either directly linked tothe DNA sequences to be expressed, or introduced into the same cell bycotransformation.

Regulatory elements required for expression include promoter sequencesto bind RNA polymerase and transcription initiation sequences forribosome binding. Additional elements may also be needed for optimalsynthesis of mRNA. These additional elements may include splice signals,as well as enhancers and termination signals. For example, a bacterialexpression vector includes a promoter such as the lac promoter and fortranscription initiation the Shine-Dalgarno sequence and the start codonAUG. Similarly, a eukaryotic expression vector includes a heterologousor homologous promoter for RNA polymerase II, a downstreampolyadenylation signal, the start codon AUG, and a termination codon fordetachment of the ribosome. Such vectors may be obtained commercially orassembled from the sequences described by methods well known in the art,for example the methods described above for constructing vectors ingeneral.

These vectors may be introduced into a suitable host cell to form a hostvector system for producing the inventive proteins. Methods of makinghost vector systems are well known to those skilled in the art.

Suitable host cells include, but are not limited to, bacterial cells(including gram positive cells), yeast cells, fungal cells, insect cellsand animal cells. Suitable animal cells include, but are not limited toHeLa cells, Cos cells, CV1 cells and various primary mammalian cells.Numerous mammalian cells may be used as hosts, including, but notlimited to, the mouse fibroblast cell NIH-3T3 cells, CHO cells, HeLacells, Ltk⁻ cells and COS cells. Mammalian cells may be transfected bymethods well known in the art such as calcium phosphate precipitation,electroporation and microinjection.

One of ordinary skill in the art will easily obtain unique sequencesfrom the cDNA cloned in the Srα plasmid. Such unique sequences may beused as probes to screen various mammalian cDNA libraries and genomicDNAs, e.g. mouse, rat and bovine, to obtain homologous nucleic acidsequences and to screen different cDNA tissue libraries to obtainisoforms of the obtained nucleic acid sequences. Nucleic acid probesfrom the cDNA cloned in the Srα plasmid may further be used to screenother human tissue cDNA libraries to obtain isoforms of the nucleic acidsequences encoding PAK4 serine/threonine kinase as well as to screenhuman genomic DNA to obtain the analogous nucleic acid sequences. Thehomologous nucleic acid sequences and isoforms may be used to producethe proteins encoded thereby.

This invention provides a plasmid comprising the nucleic acid moleculeencoding a human PAK4 serine/threonine kinase designated SrαHAPAK4 (ATCCAccession No. 209888).

This invention provides a nucleic acid probe comprising a nucleic acidmolecule of at least 15 nucleotides capable of specifically hybridizingwith a unique sequence included within the sequence of an isolatednucleic acid molecule encoding a mammalian PAK4 serine/threonine kinase.The nucleic acid probe may hybridize to any of the above-describedisolated full-length nucleic acid molecules encoding a mammalian PAK4serine/threonine kinase or fragments thereof.

This invention provides a nucleic acid probe comprising a nucleic acidmolecule of at least 15 nucleotides which is complementary to a sequenceof the isolated nucleic acid molecule encoding a mammalian PAK4serine/threonine kinase.

The nucleic acid probe is complementary to a sequence of any of theabove-described isolated full-length nucleic acid molecules encoding amammalian PAK4 serine/threonine kinase or fragments thereof.

This invention provides a method of producing a PAK4 serine/threoninekinase, which comprises growing a host cell comprising any of theabove-described vectors under suitable conditions permitting productionof the PAK4 serine/threonine kinase and recovering the PAK4serine/threonine kinase so produced. In an embodiment the method furthercomprises purifying the recovered PAK4 serine/threonine kinase.

This invention provides a method of producing a polypeptide having thebiological activity of a protein encoded by the nucleic acid moleculeencoding a PAK4 serine/threonine kinase which comprises growing theabove-described host cells under suitable conditions permittingproduction of the polypeptide and recovering the polypeptide soproduced. In another embodiment the method further comprises purifyingthe recovered polypeptide.

This invention provides a purified mammalian PAK4 serine/threoninekinase. In an embodiment the purified mammalian PAK4 serine/threoninekinase is a human PAK4 serine/threonine kinase.

This invention provides a protein comprising substantially the aminoacid sequence set forth in FIG. 1A.

This invention provides an oligonucleotide comprising a nucleic acidmolecule of at least 15 nucleotides capable of specifically hybridizingwith a unique sequence included within the sequence of the isolatednucleic acid molecule encoding a mammalian PAK4 serine/threonine kinase.In an embodiment of the oligonucleotide the nucleic acid is DNA. Inanother embodiment of the oligonucleotide the nucleic acid is RNA.

This invention provides an antisense oligonucleotide comprising asequence capable of specifically hybridizing with a unique sequenceincluded within the MRNA molecule encoding a mammalian PAK4serine/threonine kinase.

This invention provides an antisense oligonucleotide comprising asequence capable of specifically hybridizing with a unique sequenceincluded within the genomic DNA molecule encoding a mammalian PAK4serine/threonine kinase. This invention provides an antibody capable ofbinding to the PAK4 serine/threonine kinase which may be a purifiedmammalian PAK4 serine/threonine kinase or a purified human PAK4serine/threonine kinase. In an embodiment the antibody is a monoclonalantibody. In another embodiment the antibody is a polyclonal antibody.

This invention provides a monoclonal antibody directed to an epitope ofa PAK4 serine/threonine kinase.

Polyclonal antibodies may be produced by injecting a host animal such asrabbit, rat, goat, mouse or other animal with the immunogen of thisinvention, e.g. a purified mammalian PAK4 serine/threonine kinase or apurified human PAK4 serine/threonine kinase. The sera are extracted fromthe host animal and are screened to obtain polyclonal antibodies whichare specific to the immunogen. Methods of screening for polyclonalantibodies are well known to those of ordinary skill in the art such asthose disclosed in Harlow & Lane, Antibodies: A Laboratory Manual, (ColdSpring Harbor Laboratories, Cold Spring Harbor, N.Y.: 1988) the contentsof which are hereby incorporated by reference.

The monoclonal antibodies may be produced by immunizing for example,mice with an immunogen. The mice are inoculated intraperitoneally withan immunogenic amount of the above-described immunogen and then boostedwith similar amounts of the immunogen. Spleens are collected from theimmunized mice a few days after the final boost and a cell suspension isprepared from the spleens for use in the fusion.

Hybridomas may be prepared from the splenocytes and a murine tumorpartner using the general somatic cell hybridization technique ofKohler, B. and Milstein, C., Nature (1975) 256: 495-497. Availablemurine myeloma lines, such as those from the American Type CultureCollection (ATCC) 10801 University Boulevard, Manassas, Va. 20110-2209USA, may be used in the hybridization. Basically, the technique involvesfusing the tumor cells and splenocytes using a fusogen such aspolyethylene glycol. After the fusion the cells are separated from thefusion medium and grown in a selective growth medium, such as HATmedium, to eliminate unhybridized parent cells. The hybridomas may beexpanded, if desired, and supernatants may be assayed by conventionalimmunoassay procedures, for example radioimmunoassay, using theimmunizing agent as antigen. Positive clones may be characterizedfurther to determine whether they meet the criteria of the inventionantibodies.

Hybridomas that produce such antibodies may be grown in vitro or in vivousing known procedures. The monoclonal antibodies may be isolated fromthe culture media or body fluids, as the case may be, by conventionalimmunoglobulin purification procedures such as ammonium sulfateprecipitation, gel electrophoresis, dialysis, chromatography, andultrafiltration, if desired.

In the practice of the subject invention any of the above-describedantibodies may be labeled with a detectable marker. In one embodiment,the labeled antibody is a purified labeled antibody. The term “antibody”includes, by way of example, both naturally occurring and non-naturallyoccurring antibodies. Specifically, the term “antibody” includespolyclonal and monoclonal antibodies, and fragments thereof.Furthermore, the term “antibody” includes chimeric antibodies and whollysynthetic antibodies, and fragments thereof. A “detectable moiety” whichfunctions as detectable labels are well known to those of ordinary skillin the art and include, but are not limited to, a fluorescent label, aradioactive atom, a paramagnetic ion, biotin, a chemiluminescent labelor a label which may be detected through a secondary enzymatic orbinding step. The secondary enzymatic or binding step may comprise theuse of digoxigenin, alkaline phosphatase, horseradish peroxidase,β-galactosidase, fluorescein or steptavidin/biotin. Methods of labelingantibodies are well known in the art.

This invention provides a method of inhibiting PAK4 function comprisingadministering a ligand comprising an amino acid domain which binds to aGTP binding protein so as to inhibit binding of the GTP binding proteinto PAK4.

As used herein, PAK4 is a PAK4 serine/threonine kinase. As used herein,PAK4 function is defined as an effector for Cdc42Hs which mediatesinduction of filopodia.

This invention provides a method of inhibiting PAK4 function comprisingadministering a ligand which binds to the GTP binding domain of PAK4 soas to inhibit PAK4 binding to a GTP binding protein.

This invention provides a method of inhibiting PAK4 serine/threoninekinase function comprising administering a ligand which blocks an ATPbinding domain so as to inhibit PAK4 serine/threonine kinase function.

As used herein ligands comprising an amino acid domain which binds to aGTP binding protein, which binds to the GTP binding domain of PAK4, orwhich block an ATP binding domain are defined as an amino acid moleculeor fragment thereof which has an amino acid sequence complementary to aGTP binding protein, GTP binding domain of PAK4, or an ATP bindingdomain, respectively.

In an embodiment of any of the above-described methods of inhibitingPAK4 function, the inhibition of PAK4 function thereby inhibitspolymerization of actin cytoskeleton. As used herein any of theabove-described methods may be used to inhibit tumor cell growth.

In an embodiment of the method of inhibiting PAK4 function comprisingadministering a ligand comprising an amino acid domain which binds to aGTP binding protein so as to inhibit binding of the GTP binding proteinto PAK4 or the method of inhibiting PAK4 function comprisingadministering a ligand which binds to the GTP binding domain of PAK4 soas to inhibit PAK4 binding to a GTP binding protein, the GTP bindingprotein is Cdc42Hs or Rac. In an embodiment of either of these methodsthe method may further comprise inhibition of induction of filopodia.The ligand which inhibits PAK4 function may thus inhibit cytokinepathways, e.g. TNFA and growth factor pathways, e.g. epidermal growthfactor (EGF).

In an embodiment of the method of inhibiting PAK4 function comprisingadministering a ligand which binds to the GTP binding domain of PAK4 soas to inhibit PAK4 binding to a GTP binding protein or the method ofinhibiting PAK4 serine/threonine kinase function comprisingadministering a ligand which blocks an ATP binding domain so as toinhibit PAK4 serine/threonine kinase function, the ligand is an antibodycapable of binding to the PAK4 serine/threonine kinase. In anotherembodiment of these methods the antibody is a monoclonal or a polyclonalantibody.

This invention provides a method of inhibiting growth of a tumor cellcomprising blocking Cdc42Hs by administering a ligand capable of bindingto a Cdc42Hs binding site of a PAK4 serine/threonine kinase. In anembodiment of the method, the tumor cell growth is inhibited in vivo orin vitro. In another embodiment of these methods the ligand is anantibody capable of binding to the PAK4 serine/threonine kinase. In afurther embodiment of these methods the antibody is a monoclonal or apolyclonal antibody.

This invention provides a pharmaceutical composition comprising anamount of any of the above-described ligands, including, but not limitedto any of following oligonucleotides: an oligonucleotide comprising anucleic acid molecule of at least 15 nucleotides capable of specificallyhybridizing with a unique sequence included within the sequence of theisolated nucleic acid molecule encoding a mammalian PAK4serine/threonine kinase, said oligonucleotide being DNA or RNA; anantisense oligonucleotide comprising a sequence capable of specificallyhybridizing with a unique sequence included within the mRNA moleculeencoding a mammalian PAK4 serine/threonine kinase; or an antisenseoligonucleotide comprising a sequence capable of specificallyhybridizing with a unique sequence included within the genomic DNAmolecule encoding a mammalian PAK4 serine/threonine kinase, effective toprevent overexpression of a PAK4 serine/threonine kinase and apharmaceutically acceptable carrier capable of passing through a cellmembrane.

This invention provides a pharmaceutical composition comprising anamount of any of the following antibodies: an antibody capable ofbinding to the PAK4 serine/threonine kinase which may be a purifiedmammalian PAK4 serine/threonine kinase or a purified human PAK4serine/threonine kinase; said antibody being a monoclonal antibody or apolyclonal antibody; or a monoclonal antibody directed to an epitope ofa PAK4 serine/threonine kinase effective to block binding of a PAK4serine/threonine kinase to a GTP binding protein and a pharmaceuticallyacceptable carrier capable of passing through a cell membrane.

This invention provides a method of treating an abnormality in asubject, wherein the abnormality is alleviated by the inhibition ofbinding of a PAK4 serine/threonine kinase and a GTP binding proteinwhich comprises administering to the subject an effective amount of anyof the above-described pharmaceutical compositions effective to preventoverexpression of a PAK4 serine/threonine kinase, thereby treating theabnormality in the subject. In an embodiment, the GTP binding protein isCdc42Hs or Rac. In an embodiment, the abnormality is cancer orarthritis.

This invention provides a method of treating an abnormality in asubject, wherein the abnormality is alleviated by the inhibition ofbinding of a PAK4 serine/threonine kinase and a GTP binding proteinwhich comprises administering to the subject an effective amount of thepharmaceutical composition comprising any of the above-describedantibodies effective to block binding of the PAK4 serine/threoninekinase and the GTP binding protein in the subject, thereby treating theabnormality in the subject. In an embodiment, the GTP binding protein isCdc42Hs or Rac. In an embodiment, the abnormality is cancer orarthritis.

This invention provides a method of administering the above-describedpharmaceutical composition comprising an amount of any of theabove-described ligands, oligonucleotides or antibodies which aredetermined to be potentially therapeutic, wherein the administration isintravenous, intraperitoneal, intrathecal, intralymphatical,intramuscular, intralesional, parenteral, epidural, subcutaneous; byinfusion, liposome-mediated delivery, aerosol delivery; topical, oral,nasal, anal, ocular or otic delivery.

The present invention also provides a pharmaceutical compositioncomprising a effective amount of any of the above-described ligands,oligonucleotides or antibodies which are determined to be potentiallytherapeutic and a pharmaceutically acceptable carrier. In the subjectinvention an “effective amount” is any amount of the above-describedligands, oligonucleotides or antibodies which are determined to bepotentially therapeutic, which, when administered to a subject sufferingfrom a disease or abnormality against which the above-described ligands,oligonucleotides or antibodies which are determined to be potentiallytherapeutic, are effective, causes reduction, remission, or regressionof the disease or abnormality. In the practice of this invention the“pharmaceutically acceptable carrier” is any physiological carrier knownto those of ordinary skill in the art useful in formulatingpharmaceutical compositions.

In one preferred embodiment the pharmaceutical carrier may be a liquidand the pharmaceutical composition would be in the form of a solution.In another equally preferred embodiment, the pharmaceutically acceptablecarrier is a solid and the composition is in the form of a powder ortablet. In a further embodiment, the pharmaceutical carrier is a gel andthe composition is in the form of a suppository or cream. In a furtherembodiment the compound may be formulated as a part of apharmaceutically acceptable transdermal patch.

A solid carrier can include one or more substances which may also act asflavoring agents, lubricants, solubilizers, suspending agents, fillers,glidants, compression aids, binders or tablet-disintegrating agents; itcan also be an encapsulating material. In powders, the carrier is afinely divided solid which is in admixture with the finely dividedactive ingredient. In tablets, the active ingredient is mixed with acarrier having the necessary compression properties in suitableproportions and compacted in the shape and size desired. The powders andtablets preferably contain up to 99% of the active ingredient. Suitablesolid carriers include, for example, calcium phosphate, magnesiumstearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose,polyvinylpyrrolidine, low melting waxes and ion exchange resins.

Liquid carriers are used in preparing solutions, suspensions, emulsions,syrups, elixirs and pressurized compositions. The active ingredient canbe dissolved or suspended in a pharmaceutically acceptable liquidcarrier such as water, an organic solvent, a mixture of both orpharmaceutically acceptable oils or fats. The liquid carrier can containother suitable pharmaceutical additives such as solubilizers,emulsifiers, buffers, preservatives, sweeteners, flavoring agents,suspending agents, thickening agents, colors, viscosity regulators,stabilizers or osmo-regulators. Suitable examples of liquid carriers fororal and parenteral administration include water (partially containingadditives as above, e.g. cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g. glycols) and their derivatives,and oils (e.g. fractionated coconut oil and arachis oil). For parenteraladministration, the carrier can also be an oily ester such as ethyloleate and isopropyl myristate. Sterile liquid carriers are useful insterile liquid form compositions for parenteral administration. Theliquid carrier for pressurized compositions can be halogenatedhydrocarbon or other pharmaceutically acceptable propellent.

Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by for example, intramuscular, intrathecal,epidural, intraperitoneal or subcutaneous injection. Sterile solutionscan also be administered intravenously. The compounds may be prepared asa sterile solid composition which may be dissolved or suspended at thetime of administration using sterile water, saline, or other appropriatesterile injectable medium. Carriers are intended to include necessaryand inert binders, suspending agents, lubricants, flavorants,sweeteners, preservatives, dyes, and coatings.

The above-described ligands, oligonucleotides or antibodies which aredetermined to be potentially therapeutic can be administered orally inthe form of a sterile solution or suspension containing other solutes orsuspending agents, for example, enough saline or glucose to make thesolution isotonic, bile salts, acacia, gelatin, sorbitan monoleate,polysorbate 80 (oleate esters of sorbitol and its anhydridescopolymerized with ethylene oxide) and the like.

The above-described ligands, oligonucleotides or antibodies which aredetermined to be potentially therapeutic can also be administered orallyeither in liquid or solid composition form. Compositions suitable fororal administration include solid forms, such as pills, capsules,granules, tablets, and powders, and liquid forms, such as solutions,syrups, elixirs, and suspensions. Forms useful for parenteraladministration include sterile solutions, emulsions, and suspensions.

Optimal dosages to be administered may be determined by those skilled inthe art, and will vary with the particular ligands, oligonucleotides orantibodies in use, the strength of the preparation, the mode ofadministration, and the advancement of the disease condition orabnormality. Additional factors depending on the particular subjectbeing treated will result in a need to adjust dosages, including subjectage, weight, gender, diet, and time of administration.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAILS FIRST SERIES OF EXPERIMENTS MATERIALS AND METHODSIsolation of PAK4

To isolate PAK4, a pair of degenerate oligonucleotide primers weresynthesized based on the amino acid sequences conserved among the kinasedomains of yeast STE20 and human PAK65. (The two oligos corresponded tothe amino acid sequences “KKELIINE” (SEQ ID NO: 11) and “VGTPYWMA”,respectively (SEQ ID NO: 12)). The two primers were used to generate aPCR product using Jurkat cell cDNA as a template. Low stringencyconditions were used so that diverse products could be obtained. PCRproducts were gel purified and subcloned. Inserts were sequenced usingthe dideoxy chain termination method. A 400 bp PCR product containingsignificant homology to the catalytic domain of STE20 was used as aprobe to screen both a human Jurkat cell cDNA library (in the ZAPExpressTM EcoRI vector) and **a human fetal brain cDNA library (inXTriplEXTM vector). Nylon transfer filters containing 1×10⁶ recombinantplaques were hybridized with the randomly primed [α-³²P] dCTP-labeledprobe (Prime-It II kit, Stratagene) overnight at 42° C. in 6×SSC, 50%formamide, 0.1% SDS, and washed at 65° C. in 2×SSC, 0.1% SDS accordingto standard protocol. Positive plaques (≈40) were taken through furtherpurification and excised in vivo as plasmids. The positive inserts weresequenced on both strands. 5′ RACE was carried out to determine that thesequence upstream to the start codon contains an in frame stop codon.

Cell Culture and Transfection

All cells lines were grown at 37° C. in 5% CO₂ and cultured inDulbecco's modified Eagle's medium (DMEM) media containing appropriateserum (HeLa, 10% fetal bovine serum (FBS) ; NIH3T3, 10% bovine calfserum (BCS) ; COS-1, 10% newborn calf serum (NCS)). All media weresupplemented with 100 units/ml penicillin, 100 (μ/ml streptomycin, and 1mM glutamate. Transient transfection assays were carried out using thelipofectamine (GIBCO BRL) method according to the manufacturer protocol.

Protein Kinase Assays

To assay kinase activity of PAK4, NIH3T3 cells were transfected witheither empty Sra expression vector or expression vectors containing HAtagged PAK4. Cells were harvested in M2 buffer (Minden et al., 1994) 48h after transfection. Approximately 100 μg of cell extracts were mixedwith anti-HA antibody and protein A sepharose and incubated 2 h toovernight at 4° C. rotating. The immune complexes were washed two timeswith M2 buffer and two times in 20 mM HEPES, pH 7.5 and incubated inkinase buffer (described in (Minden et al., 1994)) containing, 20 μM ATPand 5 μCi of [γ-³²P]ATP either alone or together with 5 μg Histone H4(Boehringer Mannheim) or no substrate, at 30° C. for 20 min. Thereaction was stopped by boiling in 4×SDS loading buffer. Proteins wereresolved by SDS-polyacrylamide gel electrophoresis (PAGE), and substratephosphorylation and autophosphorylation were visualized byautoradiography. JNK, ERK, and p38 activity were measured as described(Minden et al., 1995).

Northern Blots

The Northern analysis was performed using a human tissue blot(Clontech). Hybridization and washes were carried out as recommended bythe manufacturer. The probe was prepared by labeling a 400 bp cDNAfragment corresponding to the kinase domain of PAK4 with [α-³²-P] dCTP(Amersham International PLC) by random priming (Stratagene).

Overlay Assay

The overlay assay is described in Martin et al., 1995. Recombinant andimmunopurified proteins were separated on SDS-PAGE followed by blottingto a PVDF membrane, washing, and blocking with PBS containing 1% BSA and100 mM DTT. Recombinant GTPases (2-5 μg) were preloaded with theindicated radiolabeled nucleotide and were incubated for 5-8 min. withthe PVDF filter. The filter was washed for 5 min. and was exposed to afilm for 2 hours. Recombinant proteins were expressed and purified fromSf9 cells as previously described (Martin et al. 1995). Various Cdc42Hseffector mutants were prepared in E. coli as GST fusion proteins andwere purified on a glutathione sepharose beads and eluted from the beadsby 5 mM reduced glutathione.

Western Blot

25 μg of COS-1 cell lysates were resolved by SDS-PAGE and transferred toPVDF membrane. The membrane was blocked in PBS containing 0.2% Tween-20(PBST) and 4% non-fat milk for 1 h and then incubated with anti HAantibody diluted in PBST containing 4% non-fat milk for 1 h. Afterwashing 3 times with PBS, the membrane was probed with secondaryantibody, peroxidase-conjugated goat IGG fraction to mouse IGG for 1 h.After washing 3 times with PBS, the immunocomplexes were visualized byenhanced chemiluminescence (ECL) reagent ECL (Amersham Corp.).

Microinjections and Immunofluorescence Microscopy

Microinjections and immunofluorescence microscopy was carried outessentially as described (Symons et al., 1996). Briefly, PAE cells weregrown in DMEM medium containing 10% fetal bovine serum and were platedon a coverslip. Expression vectors encoding various PAK4, PAK2, Cdc42Hsand Rac mutants diluted to a concentration of 50 ng/μl in injectionbuffer (5 mM glutamate, 130 mM KCL), were microinjected into the nucleusof—100 of sub-confluent PAE cells. Injected cells were incubated for16-20 hr. at 37° C. and fixed in 4% formaldehyde. Cells werepermeabilized with PBS containing 0.1% Triton x-100 and incubated in thepresence of the primary monoclonal antibodies anti HA or anti Myc for 60min. The coverslips were washed with PBS containing 0.1% Triton x-100and was incubated for 30 min. with the second antibody Texas Redconjugated anti mouse antibody. To visualized F-actin, cells were washedagain and were incubated with FITC conjugated phalloidin. Fluorescencephotomicroscopy was carried out on a Zeiss Axiophot with appropriatefilters for fluorescence detection.

Preparation of Recombinant Proteins

To prepare recombinant PAK4, a BamH1/NOTI fragment containing the fulllength PAK4 was subcloned into the pAcO-3 baculovirus expression vectorcontaining a 5′ Glu-Glu tag. Recombinant protein was then prepared andpurified as described in (Martin et al. 1995). Recombinant PAK2 wasgenerated as described in (Martin et al. 1995).

Plasmids

To construct HA tagged PAK4, an EcoRI fragment of the cDNA was ligatedin frame into the EcoRI site of Bluescript II KS(+) vector containing a5′ HA tag. HA-PAK4 was then removed from Bluescript II KS(+) as aHindIII/StuI fragment and subcloned into HindIII/SmaI site of expressionvector SRα3 (Takebe et al., 1988). PAK4(M350), PAK4(M474), andPAK4(E474) were generated by site directed mutagenesis (StratageneQuickChange kit) of K(350) or S(474) to methionine or Aspartate.Rac2L61, JNK, p38, ERK and ERK are described in Minden et al., 1995.

RESULTS Identification of PAK4, a Novel Member of the PAK Family

To identify new PAK related proteins, degenerate primers were designedcorresponding to regions of homology between the kinase domains of yeastSTE20 and mammalian hPAK2.

These primers were used to generate PCR products from Jurkat cell CDNA.The PCR products were subsequently sub-cloned and sequenced, andsequence homologies were obtained by Blast searches of the Gene Bank.Using this technique, partial sequences were identified for severalnovel putative protein kinases. One of the partial cDNAs was used as aprobe to screen Jurkat cell and fetal brain cDNA libraries as describedin materials and methods. Two identical clones that hybridize with thiscDNA were isolated. One of the clones contained a complete open readingframe of approximately 1.7 KB as shown in Figures 1A-1B. Like the PAKfamily of kinases, the carboxyl terminal portion of the predictedprotein sequence contains the 11 sub-domains that are characteristic ofserine/threonine protein kinases. Blast searches of the Gene Bankconfirmed that this region is most similar to the kinase domain of humanPAK2. This putative kinase domain has 53% identity and 73% similaritywith PAK2, and 49% identity and 71% similarity with yeast STE20 (FIG.1C). The amino terminal putative regulatory domain of PAK4 does notshare homology with any other known proteins except for a short sequenceresembling a modified GBD/CRIB domain. This conserved sequence ofapproximately 16 amino acids is found in many proteins that bind Rac andCdc42Hs (Burbelo et al., 1995). This sequence has been shown to beessential and necessary for interactions of these proteins with theGTPases (Burbelo et al., 1995). The GBD/CRIB domain found on PAK4 incomparison with those found on several other Cdc42Hs/Rac bindingproteins is shown in FIG. 1D. To determine the expression profile ofPAK4, a Northern blot was probed with a cDNA probe that corresponds tothe kinase domain of PAK4. A band of approximately 3 KB was seen in allof the human tissues that were analyzed. PAK4 appears to be most highlyexpressed in prostate, testis, and colon (FIG. 1E). A band of the samesize was detected when the Northern blot was probed with a cDNAcorresponding to the PAK4 regulatory domain (not shown).

PAK4 Interacts with GTP Bound Form of Cdc42Hs

The finding that PAK4 has a putative GBD/CRIB motif suggests that it isa new target for Cdc42Hs and/or Rac. An overlay assay was used todetermine whether PAK4 interacts with either of these GTP bindingproteins. It was found that PAK4 interacts tightly with the GTP boundform of Cdc42Hs and not with the inactive GDP bound form of Cdc42Hs. Amuch weaker interaction was detected with the GTP bound form of Rac, andno binding was observed with Rho. In contrast, PAK2 interacts withsimilar affinity to the GTP bound forms of either Rac or Cdc42Hs (FIG.2A). PAK4 lacking the GBD/CRIB domain (PAK4DGBD) does not bind toCdc42Hs, indicating that the GBD/CRIB domain is required for binding(FIG. 2B).

PAK4 Autophosphorylates and Phosphorylates an Exogenous Substrate

The analysis of PAK4's kinase activity has revealed that it behavessimilarly to other serine/threonine kinases. NIH3T3 cells weretransfected with either wild-type PAK4 or various mutants of PAK4. PAK4was immunopurif ied from cell lysates and incubated in kinase bufferwith g-³²P-ATP in the presence or absence of the substrate Histone H4.Substrate phosphorylation and autophosphorylation were analyzed afterSDS-PAGE and autoradiography. The presence of a band of the exact samesize as PAK4 strongly suggests that, like the other PAKs, PAK4 canautophosphorylate (FIG. 3A). PAK4 also co-immunopurified with a 42 kDaprotein that became phosphorylated in the kinase assay, suggesting thatit may be a substrate for PAK4 (FIG. 3A). Immunopurified PAK4 was alsoable to phosphorylate an exogenous substrate, Histone H4 (FIG. 3B). Theactivity of a PAK4(M350) mutant was also tested. This mutant contains amutation in which the conserved lysine in subdomain II is converted to anon-phosphorylatable residue, methionine. Mutation of this conservedlysine disrupts the ATP binding site of nearly all serine/threoninekinases that have been analyzed (Hanks et al., 1988). ImmunopurifiedPAK4 (M350) was completely unable to autophosphorylate or, as shown inFIG. 3B, to phosphorylate Histone H4. Most serine/threonine kinasescontain a conserved serine or threonine residue within the linker regionbetween kinase subdomains VII and VIII. This residue usually becomesphosphorylated either by autophosphorylation or by an upstream kinase.Phosphorylation of this residue is essential for the activities of mostserine/threonine kinases (Johnson et al., 1996; Marshall, 1994; Pelech,1996; Zhang et al., 1994). Consistent with this, mutation of thecorresponding serine in PAK4 to a methionine results in a completelyinactive PAK4 (M474) (FIG. 3B). Many protein kinases become furtheractivated when the regulatory domain is removed. PAK4D which containsonly the kinase domain was generated. PAK4D exhibits at least 5 foldmore kinase activity than wild-ype PAK4, suggesting that the carboxylterminal portion of PAK4 contains a regulatory domain (FIG. 3B). Likethe wild-type protein however, mutation of the lysine in subdomain II togenerate PAK4D(M350) results in a completely inactive kinase (FIG. 3B).

PAK4 Activates the JNK Pathway

One of the functions of Cdc42Hs and Rac is to activate the JNK and p38MAP Kinase pathways (Bagrodia et al., 1995; Brown et al., 1996; Coso etal., 1995; Minden et al., 1995; Zhang et al., 1995). Since PAK4 is atarget for Cdc42Hs, its ability to activate mammalian MAP Kinasepathways was tested. NIH3T3 cells were transfected with increasing dosesof an expression vector containing the PAK4 cDNA. These wereco-transfected with expression vectors containing epitope tagged JNK,ERK, or p38 cDNAs. After transient expression, JNK, ERK, or p38 wereimmunopurified from cell lysates using antibodies against the epitopetag, followed by an in vitro kinase assay. The results indicate thatoverexpression of the wild-type PAK4 leads to activation of the JNKpathway, although this activation is somewhat weak compared with otherJNK activators such as Rac or Cdc42Hs (FIG. 3C). PAK4 has littleactivity towards ERK, and does not activate the p38 pathway (FIGS. 3Dand E).

PAK4 induces localized actin polymerization and induces the formation offilopodia

Since PAK4 is a novel target for Cdc42Hs, its role in the induction ofcytoskeletal changes was studied. Microinjection of fibroblasts withpurified constitutively active Cdc42HsV12 protein has been shown to leadto a transient induction of filopodia (Kozma et al., 1995; Nobes andHall, 1995). The filopodia disappear within a short time (by 15-30minutes) after microinjection of the protein followed by the formationof lamellipodia, due to the ability of Cdc42Hs to activate Rac (Kozma etal., 1995; Nobes and Hall, 1995). Another consequence of Cdc42Hsmicroinjection is the dissolution of stress fibers. To see whether PAK4can potentiate the cytoskeletal effects triggered by the GTPases, PAEcells were microinjected with expression vectors for HA tagged PAK4either alone or together with myc tagged Cdc42HsV12 or Rac1V12expression vectors (Symons et al., 1996). The experiments describedherein were also carried out with NIH3T3 fibroblasts, with similarresults. As shown in FIGS. 4A-4F, when expressed alone, PAK4 waslocalized in a perinuclear area and did not affect the actincytoskeleton. Cells microinjected with expression vectors forconstitutively active Rac1V12 or Cdc42HsV12 alone induced extensivelamellipodia. Filopodia were not observed in the Cdc42Hs injected cellsbecause cells were analyzed between 12-16 hours after microinjection.Interestingly, co-expression of PAK4 with Cdc42HsV12 caused dramaticchanges in the actin cytoskeleton and re-distribution of PAK4. Whenco-injected with Cdc42HsV12, PAK4 became concentrated in one side of thenucleus in an area that resembles the trans Golgi compartment (FIGS.4A-4F). Dual staining with phalloidin and HA antibody revealed astriking co-localization of PAK4 with polymerized actin clusters (FIGS.4A-4F), and in cells expressing lower levels of PAK4, polymerized actinwas detected around vesicles (data not shown). In addition,co-expression of PAK4 and Cdc42HsV12 induced the sustained formation ofactin enriched filopodia protrusions in more than 70% of the injectedcells and also caused the dis-assembly of stress fibers (FIGS. 4A-4F).Co-expression of PAK4 with a dominant negative Cdc42HsN17 had no effecton PAK4 localization or the reorganization of the actin cytoskeleton(data not shown). Co-expression of PAK4 and the constitutively activeRac1V12 resulted in a very similar phenotype to that observed withRac1V12 alone. Rac1V12 induced the formation of lamellipodia andrelocalization of a small percentage of PAK4 from the perinuclear areato the lamellipodia (FIGS. 4A-4F) . To test whether these phenotypes arespecific for PAK4, PAK2 expression vector with either Rac1V12 orCdc42HsV12 expression vector was co-injected. PAK2 was localized in thecytosol and the nucleus and was not redistributed by Rac or Cdc42Hs(data not shown). In addition, cells co-expressing Rac1V12 or Cdc42HsV12together with PAK2 had similar cytoskeletal phenotypes as cellsexpressing Rac1V12 or Cdc42Hs alone (FIGS. 4G-4J). This data stronglysuggests that in contrast to PAK2, PAK4 is the effector for Cdc42Hs thatleads to the induction of filopodia and actin polymerization.

PAK4 is Recruited to the Golgi Apparatus by Activated Cdc42Hs

PAK4 appears to be regulated by specific recruitment by Cdc42Hs to anarea resembling the trans-Golgi. It is interesting to note that theendogenous Cdc42Hs was recently shown to be localized primarily at Golgimembranes, and to localize with the Golgi membrane coatomer proteinb-COP (Erickson et al., 1996). In addition, a recently identifiedCdc42Hs related protein was also localized to the Golgi structure(unpublished observation). To determine whether PAK4 also localizes tothe Golgi, injected cells were co-stained with anti HA and anti b-COPantibody. As presented in FIGS. 5A-5H, in cells expressing PAK4 alone,PAK4 was localized to perinuclear areas and was not co-localized withb-COP. In cells co-expressing PAK4 and Cdc42HsV12 however, PAK4 wasco-localized with b-COP in the Golgi.

The effects of the drug Brefeldin A (BFA) on PAK4 location were tested.Golgi coatomer proteins are normally redistributed when cells areexposed to BFA (Orci et al., 1991). Likewise, Cdc42Hs is redistributedfrom its Golgi location when cells are treated with BFA (Erickson etal., 1996). Interestingly, treatment of injected cells with BFA wasfound to also cause a rapid redistribution of PAK4 from the Golgi to thecytoplasm and the nucleus. This is nearly identical to the effects ofBFA on b-COP in these cells (FIGS. 5A-5H). This data strongly suggeststhat in the presence of Cdc42Hs, PAK4 is localized at a BFA sensitivecomponent of Golgi membranes. In addition, BFA treated cells containedless filopodia and polymerized actin. This data suggests that thelocalization of PAK4 by Cdc42Hs to the Golgi may play an important rolein the reorganization of actin.

PAK4 Kinase Activity is Required for its Ability to Induce LocalizedActin Polymerization and to Potentiate Filopodia Formation by Cdc42Hs

The effect of PAK4 mutants on the formation of filopodia and actinpolymerization was tested. The kinase inactive PAK4(M350) wasmicroinjected into cells either alone or together with Cdc42HsV12. LikePAK4 wt, PAK4(M350) was localized in the perinuclear area and wasrecruited by Cdc42Hs to the BFA sensitive compartment of the Golgi(FIGS. 6A-6H). However, in contrast to PAK4 wt, PAK4(M350) was unable toinduce localized actin polymerization or the formation of filopodia, anddid not lead to a reduction in stress fibers (FIGS. 6A-6H) . A putativeconstitutively active PAK4 mutant was also generated. This mutant wasgenerated by mutation of serine 474 (in the linker region betweensubdomains VII and VIII) to a glutamic acid. The resulting PAK(E474)mutant had a greatly enhanced autophosphorylation activity (data notshown). Mutation of this conserved site to a negatively charged aminoacid has been found to generate constitutively active mutants of manyserine/threonine kinases including the PAKs (Benner et al., 1995; Manseret al., 1997; Szczepanowska et al., 1997). When expressed alone,PAK4(E474) localized to similar areas as PAK4 wt and PAK4(M350), and didnot have any effect on the actin cytoskeleton. Like PAK4 wt, PAK4(E474)was recruited specifically by Cdc42HsV12 to the Golgi and induced theformation of filopodia and polymerization of actin to the same extent aswild-type PAK4 (FIGS. 6A-6H). Taken together, these data indicate thatPAK4's kinase activity is not necessary for the cellular localization ofPAK4, but is essential for its effects on actin polymerization. Thekinase activity of wild-type PAK4 appears to be sufficient to induce thecytoskeletal changes however, as the PAK4(E474) mutant had no enhancedeffect on actin polymerization and filopodia formation. Furthermore,unlike results described for PAK1, constitutively active PAK4 did notinduce cytoskeletal changes on its own, but maintained the requirementfor recruitment by Cdc42Hs to the golgi.

PAK4 Binds Cdc42Hs Effector Mutants to Induce Filopodia and ActinPolymerization

Two effector mutants of constitutively active Cdc42HsL61 were previouslyexamined to assess the role of PAK and other effectors in filopodiaformation (Lamarche et al., 1996). Both mutants had single amino acidsubstitutions in the effector loop. One mutant, Cdc42HsL61(Y40C) couldnot bind PAK or several other GBD/CRIB domain containing proteins. Theother mutant, Cdc42HsL61(F37A), maintained the ability to bind PAK andother GBD/CRIB domain containing proteins. Interestingly, both effectormutants were equally efficient in the ability to induce filopodia whenmicroinjected into fibroblasts together with dominant negative Rac(Lamarche et al., 1996). These results would tend to suggest that PAKbinding to Cdc42Hs is not necessary for filopodia formation. To seewhether Cdc42HsL61(Y40C) could be mediating its effects through PAK4,PAK4 binding to Cdc42HsL61 and to the two effector mutants was tested.For comparison PAK2 and another GBD/CRIB containing protein, WASP(Symons et al., 1996) were also analyzed. As shown in FIG. 7A, all threeproteins bound to Cdc42HsL61. Likewise, as expected, they also all boundto Cdc42Hs(F37A), though with a slightly lower affinity. Surprisinglyhowever, PAK4 bound Cdc42HsL61(Y40C) with a similar affinity as wasdetected with Cdc42HsL61(F37A), although WASP and PAK2 did not bindCdc42HsL61(Y40C) efficiently. Moreover, when Cdc42HsL61(C40) wasco-injected into PAE cells with PAK4, PAK4 was recruited to the Golgiarea, induced actin polymerization, and promoted the formation offilopodia (see FIGS. 7B-7C) The results suggest a mechanism whereby theeffector mutant Cdc42HsL61(Y40C) can induce filopodia formation.Although it can not efficiently bind PAK2, this mutant maintains theability to interact with PAK4. Importantly, it was found that PAK4,rather than the other known PAKs, is the important mediator of filopodiaformation by Cdc42Hs.

DISCUSSION

PAK4 was identified as a novel member of the PAK family. Like the otherPAKs, PAK4 contains a GBD/CRIB domain at the N-terminus and a kinasedomain at the C-terminus. The overall sequence identity to other PAKs,however, is significantly different. Although the kinase domain of PAK4is more similar in sequence to the kinase domains of PAK 1, 2, and 3than to any other known proteins, it shares only 53% sequence identity.PAK4 exhibits no sequence homology in the regulatory domain outside theGBD/CRIB sequences. Even the GBD/CRIB motif is similar, but notidentical, to the GBD/CRIB motif in the other PAKs. This suggests thatPAK4 may have a different function than the known PAKs. It was shownthat PAK4 interacts preferentially with the GTP bound form of Cdc42Hsand activates the JNK family of MAP Kinases. Moreover, microinjection ofPAK4 and Cdc42Hs plasmids into different cell types demonstrates thatPAK4 has a profound effect on the actin cytoskeleton. When co-injectedwith Cdc42Hs, PAK4 is recruited by Cdc42Hs to the brefeldin A sensitivecompartment of the Golgi, and subsequently induces actin polymerizationat the Golgi. Strikingly, PAK4 also greatly stimulates the induction offilopodia when it is co-injected with Cdc42Hs. In contrast to otherPAKs, PAK4 kinase activity and its interaction with the activatedCdc42Hs are essential for the induction of this phenotype. In addition,PAK4 interacts with the Cdc42Hs effector mutant (Cdc42L61(C40)) whichwas previously shown to be important in the induction of filopodia butwhich failed to bind other PAKs (Lamarche et al., 1996). The dataprovides a novel link between Cdc42Hs, PAK4, and actin polymerization,and supports the idea that the Golgi apparatus plays a role incytoskeletal re-organization.

The Rho family of GTPases play key roles in the control of cellmorphology. By using microinjection techniques it was demonstrated thatCdc42Hs triggers the formation of microspikes and filopodia followed byactivation of Rac, which leads to the formation of lamellipodia. A thirdGTPase, RhoA, is implicated in the formation of stress fibers. Inaddition, all three GTPases regulate the assembly of focal complexes.The cytoskeletal changes triggered by these GTPases play important rolesin cell motility and division and in the maintenance of cell shape. Theidentification of molecular targets which mediate these cytoskeletaleffects is therefore of great importance. Several proteins were shown tointeract with the activated form of Cdc42Hs, however, the moleculareffector that links Cdc42Hs to the formation of filopodia has not yetbeen identified. Recent experiments with various effector mutants ofCdc42Hs demonstrated that the cytoskeletal changes induced by Cdc42Hsare independent of PAK1, 2 or 3. In this report it has been shown that anovel PAK related protein, PAK4, interacts only with the GTP bound formof Cdc42Hs and not with other Rho members. The interaction betweenCdc42Hs and PAK4 is essential for targeting PAK4 to the Golgicompartment and subsequently for the re-organization of the actincytoskeleton. In contrast to PAK1, 2, and 3, the kinase activity of PAK4is not regulated by Cdc42Hs (data not shown). Thus, it appears to berecruitment of PAK4 by Cdc42Hs, rather than stimulation of its kinaseactivity, which is important for actin polymerization and cytoskeletalchanges. PAK4's kinase activity is necessary however, because a kinaseinactive PAK4 mutant was re-localized to the Golgi by Cdc42Hs, butfailed to reorganize the actin cytoskeleton. In addition to kinaseactivity, localization by Cdc42Hs is critical for PAK4 to induce actinpolymerization and filopodia formation. Even a constitutively activePAK4 could not bypass the need for recruitment by Cdc42Hs to the Golgi.This mutant localized to the same area in the cell as wild-type PAK4,and only when it was recruited to the Golgi compartment by Cdc42Hs didinduce the formation of filopodia and actin polymerization. Theseexperiments strongly suggest that PAK4 mediates its biological effect bytranslocation to a specific site by Cdc42Hs and thereby bringing PAK4 toclose proximity with a putative substrate.

This mechanism of activation of PAK4 is substantially different than theone proposed for other PAK members. PAK 1 was shown to be recruited tothe focal complexes induced by Cdc42Hs, but the recruitment to thesesites did not require a direct interaction with Cdc42Hs. Recently, itwas demonstrated that PAK1 is recruited to the focal complexes byinteracting with a novel GTP/GDP exchange factor PIX. A specific prolinerich sequence on the PAK regulatory domain interacts with the SH3 domainof PIX. Furthermore, it was proposed that the PAK/PIX complex activatesthe Rac signaling pathways. Interestingly, PAK kinase activity was notrequired for activation of the Rac pathway, but was shown to beimportant in the disassembly of the focal complexes. PAK1 has also beenreported to induce filopodia and lamellipodia similar to those inducedby Cdc42Hs and Rac. However, these cytoskeletal changes occurindependently of PAK1's ability to bind Cdc42Hs and Rac, and are partlyindependent of its kinase activity. These results, coupled with theresults from experiments using effector mutants of Rac and Cdc42Hssuggest that, while PAK1 may be able to induce cytoskeletal changes whenit is overexpressed, it might not be the link between GTPases and thecytoskeleton.

Recent studies indicated that Cdc42Hs is localized to the BFA sensitivecompartment of the Golgi apparatus. The role of Cdc42Hs in the Golgi ispoorly understood. The Golgi apparatus is known mostly for its roles inthe formation of transport vesicles which carry cargo to a receivingcompartment. Nonclathrin coat proteins such as bCOP and the GTPase ARFwere shown to be implicated in Golgi mediated transport. Disruption ofthe coatomer complexes by treating cells with BFA or expressing dominantnegative forms of ARF blocked the transport of vesicles to the plasmamembrane. These data indicated that PAK4 is co-localized with bCOP andis redistributed by treating the cells with BFA. Furthermore, BFAtreatment affected the filopodia formation and actin polymerizationinduced by PAK4 and Cdc42HsV12. Interestingly, it was previouslyreported that fibroblast treated with BFA failed to make filopodia andlamellipodia and subsequently cells were defective in motility. It hasbeen proposed that the disruption of the Golgi apparatus by BFA mayaffect the supply of vesicles containing the components necessary forthe formation of filopodia. Because it is localized by Cdc42Hs to theGolgi, PAK4 is a good candidate for an effector molecule that transducesa Cdc42Hs dependent signal from the Golgi. It is conceivable that PAK4and Cdc42Hs regulate the re-organization of actin and the formation offilopodia by controlling the transport of vesicles containing theproteins and/or lipids necessary for the induction of morphologicalchanges. Interestingly, in addition to the induction of filopodia, PAK4also leads to localized actin polymerization at the Golgi area. Infuture studies it will be interesting to determine whether actinpolymerization in this area is important for vesicle fusion or otheraspects of Golgi function.

In summary, PAK4 provides a molecular link between Cdc42Hs and actinrearrangement, and suggests involvement of the Golgi apparatus in cellmorphogenesis. Understanding how PAK4 together with Cdc42Hs and putativetargets at the Golgi control the reorganization of the actincytoskeleton will contribute to the understanding of the molecularmechanism of morphogenesis.

SECOND SERIES OF EXPERIMENTS Mouse PAK4 Sequences

1) Partial cDNA of the Mouse PAK4:

AAGCAGCAGC GGCGCGAGTT GCTCTTCAAT GAGGTGGTGA TCATGCGGGA (SEQ ID NO: 13)CTACCGGCAC GAGAACGTGG TGGAGATGTA CAACAGCTAC CTGGTGGGTG ACGAACTCTGGGTCGTCATG GAGTTCCTGG AAGGCGGCGC CCTCACGGAT ATTGTCACCC ACACCAGGATGAACGAGGAA CAGATCGCCG CCGTGTGCCT GGCTGTGCTT CAGGCGCTGG CTGTGCTCCACGCCCAGGGT GTCATCCACA GCGACATAAA AACGGACA

2) Predicted Amino Acid Sequence of the Partial Mouse PAK4 cDNA:

KQQRRELLFN EVVIMRDYRH ENVVEMYNSY LVGDELWVVM EFLEGGALTD (SEQ ID NO: 14)IVTHTRMNEE QIAAVCLAVL QALAVLHAQG VIHSDIKTD

3) Partial Genomic Sequence of Mouse PAK4:

ACCTGGTGGG TGACGAACTC TGGGTCGTCA TGGAGTTCCT GGAAGGCGGC (SEQ ID NO: 15)GCCCTCACGG ATATTGTCAC CCACACCAGG TACCATAGGG CAGCCTGCTG GCTCATGTGCTCCCTGGGGT GGAACTGGGA CCCTTTAGGC TCTGGTGATA GACAAGTGCC CTCCAGAGTGTGGGTGGGGC AGTGAGGCCA GGCACACAGG ATGGGGGTCA TAGCATCGTG GCTCCCTGACCCCTGTTGAG GCGGGTCTTT GTGACCTCTT GTTGTCTAAA GCAGGGTAGG GGCCTCTTCACTGCCCACTC TCACCCCAGG GTGGGATGCC CAAGGCAGCG CTGAGTGCCC AGTTGCTCCTCTGCCCGCGC AGGATGAACG AGGAACAGAT CGCCGCCCGT GTGCCTGGCT TGTGCTTCANGCGCTGGCTT GTGCTCCACG CCCAGGGTGT CATCCACCGT GACATCAAGA GTGACTCTATCTTGCTGACC CATGATGGC

METHODS

1) Isolation of the Partial Mouse CDNA:

The partial mouse cDNA was generated by degenerate PCR using degenerateprimers corresponding to the following amino acid sequences: (a) GEGSTG(SEQ ID NO: 16) (b) SLVGTP (SEQ ID NO: 17) within the kinase domain ofPAK4. The degenerate primers were used in a PCR reaction using mousecDNA (from NIH3T3 cells) as a template.

2) Isolation of the Full Length Mouse PAK4;

The paertial cDNA is currently being used to isolate the full lengthPAK4cDNA. Th epartial cDNA will be used as a probe to screen a mousebrain cDNA library (Stratagene). Nylon transfer filters containing 1×10⁶recombinant plaques will be hybridized with the randomlyprimed [α-32P]dCTP-labeled probe (Prime-It II kit, Stratagene) overnight at 42° C. in6×SSC, 50% formamide, 0.1% SDS and washed at 65° C. in 2×SSC, 0.1% SDSaccording to standard protocol. Positive plaques will be taken throughfurther purification and excised in vivo as plasmids. The positiveinserts will be sequenced in both strands. If necessary, 5′ RACE and 3′RACE will be carried out to isolate the 5′ and 3′ ends.

3) Isolation of the Mouse Genomic Sequence:

The partial mouse CDNA was used as a probe to screen a BAC system(Bacterial Artificial Chromosome) mouse genomic library (GenomeSystems). Positive clones were subcloned into a Bluescript vector andanalyzed by Southern Blots and sequencing. One of the positive clones (8kb) has been sequenced partially, and found to contain the sequenceshown above. This clone will be further sequenced to determine whetherit contatins the full length genomic PAK4 sequence, it will be used as aprobe to undergo a further round of screening, until the full lengthsequence is isolated.

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17 1 1940 DNA human 1 tgagggaggc gcgagggcgc ggagttccag gtcgagcagttaggccgcga gcgactgcgg 60 cgccgagccg atgagtaacc cgaagcccct agaggagtggtcacctgcct gagggcactt 120 ctgtcccacc agcatcagac caggccgcac cgagtccccggcaccatgtt tgggaagagg 180 aagaagcggg tggagatctc cgcgccgtcc aacttcgagcaccgcgtgca cacgggcttc 240 gaccagcacg agcagaagtt cacggggctg ccccgccagtggcagagcct gatcgaggag 300 tcggctcgcc ggcccaagcc cctcgtcgac cccgcctgcatcacctccat ccagcccggg 360 gcccccaaga ccatcgtgcg gggcagcaaa ggtgccaaagatggggccct cacgctgctg 420 ctggacgagt ttgagaacat gtcggtgaca cgctccaactccctgcggag agacagcccg 480 ccgccgcccg cccgtgcccg ccaggaaaat gggatgccagaggagccggc caccacggcc 540 agagggggcc cagggaaggc aggcagccga ggccggttcgccggtcacag cgaggcaggt 600 ggcggcagtg gtgacaggcg acgggcgggg ccagagaagaggcccaagtc ttccagggag 660 ggctcagggg gtccccagga gtcctcccgg gacaaacgccccctctccgg gcctgatgtc 720 ggcacccccc agcctgctgg tctggccagt ggggcgaaactggcagctgg ccggcccttt 780 aacacctacc cgagggctga cacggaccac ccatcccggggtgcccaggg ggagcctcat 840 gacgtggccc ctaacgggcc atcagcgggg ggcctggccatcccccagtc ctcctcctcc 900 tcctcccggc ctcccacccg agcccgaggt gcccccagccctggagtgct gggaccccac 960 gcctcagagc cccagctggc ccctccagcc tgcacccccgccgcccctgc tgttcctggg 1020 ccccctggcc cccgctcacc acagcgggag ccacagcgagtatcccatga gcagttccgg 1080 gctgccctgc agctggtggt ggacccaggc gacccccgctcctacctgga caacttcatc 1140 agattggcga gggctccacg ggcatcgtgt gcatcgccaccgtgcgcagc tcgggcaagc 1200 tggtggccgt caagaagatg gacctgcgca agcagcagaggcgcgagctg ctcttcaacg 1260 aggtggtaat catgagggac taccagcacg agaatgtggtggagatgtac aacagctacc 1320 tggtggggga cgagctctgg gtggtcatgg agttcctggaaggaggcgcc ctcaccgaca 1380 tcgtcaccca caccaggatg aacgaggagc agatcgcggccgtgtgcctt gcagtgctgc 1440 aggccctgtc ggtgctccac gcccagggcg tcatccaccgggacatcaag agcgactcga 1500 tcctgctgac ccatgatggc agggtgaagc tgtcagactttgggttctgc gcccaggtga 1560 gcaaggaagt gccccgaagg aagtcgctgg tcggcacgccctactggatg gccccagagc 1620 tcatctcccg ccttccctac gggccagagg tagacatctggtcgctgggg ataatggtga 1680 ttgagatggt ggacggagag cccccctact tcaacgagccacccctcaaa gccatgaaga 1740 tgattcggga caacctgcca ccccgactga agaacctgcacaaggtgtcg ccatccctga 1800 agggcttcct ggaccgcctg ctggtgcgag accctgcccagcgggccacg gcagccgagc 1860 tgctgaagca cccattcctg gccaaggcag ggccgcctgccagcatcgtg cccctcatgc 1920 gccagaaccg caccagatga 1940 2 591 PRT human 2Met Phe Gly Lys Arg Lys Lys Arg Val Glu Ile Ser Ala Pro Ser Asn 1 5 1015 Phe Glu His Arg Val His Thr Gly Phe Asp Gln His Glu Gln Lys Phe 20 2530 Thr Gly Leu Pro Arg Gln Trp Gln Ser Leu Ile Glu Glu Ser Ala Arg 35 4045 Arg Pro Lys Pro Leu Val Asp Pro Ala Cys Ile Thr Ser Ile Gln Pro 50 5560 Gly Ala Pro Lys Thr Ile Val Arg Gly Ser Lys Gly Ala Lys Asp Gly 65 7075 80 Ala Leu Thr Leu Leu Leu Asp Glu Phe Glu Asn Met Ser Val Thr Arg 8590 95 Ser Asn Ser Leu Arg Arg Asp Ser Pro Pro Pro Pro Ala Arg Ala Arg100 105 110 Gln Glu Asn Gly Met Pro Glu Glu Pro Ala Thr Thr Ala Arg GlyGly 115 120 125 Pro Gly Lys Ala Gly Ser Arg Gly Arg Phe Ala Gly His SerGlu Ala 130 135 140 Gly Gly Gly Ser Gly Asp Arg Arg Arg Ala Gly Pro GluLys Arg Pro 145 150 155 160 Lys Ser Ser Arg Glu Gly Ser Gly Gly Pro GlnGlu Ser Ser Arg Asp 165 170 175 Lys Arg Pro Leu Ser Gly Pro Asp Val GlyThr Pro Gln Pro Ala Gly 180 185 190 Leu Ala Ser Gly Ala Lys Leu Ala AlaGly Arg Pro Phe Asn Thr Tyr 195 200 205 Pro Arg Ala Asp Thr Asp His ProSer Arg Gly Ala Gln Gly Glu Pro 210 215 220 His Asp Val Ala Pro Asn GlyPro Ser Ala Gly Gly Leu Ala Ile Pro 225 230 235 240 Gln Ser Ser Ser SerSer Ser Arg Pro Pro Thr Arg Ala Arg Gly Ala 245 250 255 Pro Ser Pro GlyVal Leu Gly Pro His Ala Ser Glu Pro Gln Leu Ala 260 265 270 Pro Pro AlaCys Thr Pro Ala Ala Pro Ala Val Pro Gly Pro Pro Gly 275 280 285 Pro ArgSer Pro Gln Arg Glu Pro Gln Arg Val Ser His Glu Gln Phe 290 295 300 ArgAla Ala Leu Gln Leu Val Val Asp Pro Gly Asp Pro Arg Ser Tyr 305 310 315320 Leu Asp Asn Phe Ile Lys Ile Gly Glu Gly Ser Thr Gly Ile Val Cys 325330 335 Ile Ala Thr Val Arg Ser Ser Gly Lys Leu Val Ala Val Lys Lys Met340 345 350 Asp Leu Arg Lys Gln Gln Arg Arg Glu Leu Leu Phe Asn Glu ValVal 355 360 365 Ile Met Arg Asp Tyr Gln His Glu Asn Val Val Glu Met TyrAsn Ser 370 375 380 Tyr Leu Val Gly Asp Glu Leu Trp Val Val Met Glu PheLeu Glu Gly 385 390 395 400 Gly Ala Leu Thr Asp Ile Val Thr His Thr ArgMet Asn Glu Glu Gln 405 410 415 Ile Ala Ala Val Cys Leu Ala Val Leu GlnAla Leu Ser Val Leu His 420 425 430 Ala Gln Gly Val Ile His Arg Asp IleLys Ser Asp Ser Ile Leu Leu 435 440 445 Thr His Asp Gly Arg Val Lys LeuSer Asp Phe Gly Phe Cys Ala Gln 450 455 460 Val Ser Lys Glu Val Pro ArgArg Lys Ser Leu Val Gly Thr Pro Tyr 465 470 475 480 Trp Met Ala Pro GluLeu Ile Ser Arg Leu Pro Tyr Gly Pro Glu Val 485 490 495 Asp Ile Trp SerLeu Gly Ile Met Val Ile Glu Met Val Asp Gly Glu 500 505 510 Pro Pro TyrPhe Asn Glu Pro Pro Leu Lys Ala Met Lys Met Ile Arg 515 520 525 Asp AsnLeu Pro Pro Arg Leu Lys Asn Leu His Lys Val Ser Pro Ser 530 535 540 LeuLys Gly Phe Leu Asp Arg Leu Leu Val Arg Asp Pro Ala Gln Arg 545 550 555560 Ala Thr Ala Ala Glu Leu Leu Lys His Pro Phe Leu Ala Lys Ala Gly 565570 575 Pro Pro Ala Ser Ile Val Pro Leu Met Arg Gln Asn Arg Thr Arg 580585 590 3 250 PRT human 3 Phe Ile Lys Ile Gly Glu Gly Ser Thr Gly IleVal Cys Ile Ala Thr 1 5 10 15 Val Arg Ser Ser Gly Lys Leu Val Ala ValLys Lys Met Asp Leu Arg 20 25 30 Lys Gln Gln Arg Arg Glu Leu Leu Phe AsnGlu Val Val Ile Met Arg 35 40 45 Asp Tyr Gln His Glu Asn Val Val Glu MetTyr Asn Ser Tyr Leu Val 50 55 60 Gly Asp Glu Leu Trp Val Val Met Glu PheLeu Glu Gly Gly Ala Leu 65 70 75 80 Thr Asp Ile Val Thr His Thr Arg MetAsn Glu Glu Gln Ile Ala Ala 85 90 95 Val Cys Leu Ala Val Leu Gln Ala LeuSer Val Leu His Ala Gln Gly 100 105 110 Val Ile His Arg Asp Ile Lys SerAsp Ser Ile Leu Leu Thr His Asp 115 120 125 Gly Arg Val Lys Leu Ser AspPhe Gly Phe Cys Ala Gln Val Ser Lys 130 135 140 Glu Val Pro Arg Arg LysSer Leu Val Gly Thr Pro Tyr Trp Met Ala 145 150 155 160 Pro Glu Leu IleSer Arg Leu Pro Tyr Gly Pro Glu Val Asp Ile Trp 165 170 175 Ser Leu GlyIle Met Val Ile Glu Met Val Asp Gly Glu Pro Pro Tyr 180 185 190 Phe AsnGlu Pro Pro Leu Lys Ala Met Lys Met Ile Arg Lys Asn Leu 195 200 205 ProPro Arg Leu Lys Asn Leu His Lys Val Ser Pro Ser Leu Lys Gly 210 215 220Phe Leu Asp Arg Leu Leu Val Arg Asp Pro Ala Gln Arg Ala Thr Ala 225 230235 240 Ala Glu Leu Leu Lys His Pro Phe Leu Ala 245 250 4 250 PRT human4 Tyr Glu Lys Ile Gly Gln Gly Ala Ser Gly Thr Val Phe Thr Ala Thr 1 5 1015 Asp Val Ala Leu Gly Gln Glu Val Ala Ile Lys Gln Ile Asn Leu Gln 20 2530 Lys Gln Pro Lys Lys Glu Leu Ile Ile Asn Glu Ile Leu Val Met Lys 35 4045 Glu Leu Lys Asn Pro Asn Ile Val Asn Phe Leu Asp Ser Tyr Leu Val 50 5560 Gly Asp Glu Leu Phe Val Val Met Glu Tyr Leu Ala Gly Arg Ser Leu 65 7075 80 Thr Asp Val Val Thr Glu Thr Cys Met Asp Glu Ala Gln Ile Ala Ala 8590 95 Val Cys Arg Glu Cys Leu Gln Ala Leu Glu Phe Leu His Ala Asn Gln100 105 110 Val Ile His Arg Asp Ile Lys Ser Asp Asn Val Leu Leu Gly MetGlu 115 120 125 Gly Ser Val Lys Leu Thr Asp Phe Gly Phe Cys Ala Gln IleThr Pro 130 135 140 Glu Gln Ser Lys Arg Ser Thr Met Val Gly Thr Pro TyrTrp Met Ala 145 150 155 160 Pro Glu Val Val Thr Arg Lys Ala Tyr Gly ProLys Val Asp Ile Trp 165 170 175 Ser Leu Gly Ile Met Ala Ile Glu Met ValGlu Gly Glu Pro Pro Tyr 180 185 190 Leu Asn Glu Asn Pro Leu Arg Ala LeuTyr Leu Ile Ala Thr Asn Gly 195 200 205 Thr Pro Glu Leu Gln Asn Pro GluLys Leu Ser Pro Ile Phe Arg Asp 210 215 220 Phe Leu Asn Arg Cys Leu GluMet Asp Val Glu Lys Arg Gly Ser Ala 225 230 235 240 Lys Glu Leu Leu GlnHis Pro Phe Leu Lys 245 250 5 250 PRT yeast 5 Leu Val Lys Ile Gly GlnGly Ala Ser Gly Gly Val Tyr Thr Ala Tyr 1 5 10 15 Glu Ile Gly Thr AsnVal Ser Val Ala Ile Lys Gln Met Asn Leu Glu 20 25 30 Lys Gln Pro Lys LysGlu Leu Ile Ile Asn Glu Ile Leu Val Met Lys 35 40 45 Gly Ser Lys His ProAsn Ile Val Asn Phe Ile Asp Ser Tyr Val Leu 50 55 60 Lys Gly Asp Leu TrpVal Ile Met Glu Tyr Met Glu Gly Gly Ser Leu 65 70 75 80 Thr Asp Val ValThr His Cys Ile Leu Thr Glu Gly Gln Ile Gly Ala 85 90 95 Val Cys Arg GluThr Leu Ser Gly Leu Glu Phe Leu His Ser Lys Gly 100 105 110 Val Leu HisArg Asp Ile Lys Ser Asp Asn Ile Leu Leu Ser Met Glu 115 120 125 Gly AspIle Lys Leu Thr Asp Phe Gly Phe Cys Ala Gln Ile Asn Glu 130 135 140 LeuAsn Leu Lys Arg Thr Thr Met Val Gly Thr Pro Tyr Trp Met Ala 145 150 155160 Pro Glu Val Val Ser Arg Lys Glu Tyr Gly Pro Lys Val Asp Ile Trp 165170 175 Ser Leu Gly Ile Met Ile Ile Glu Met Ile Glu Gly Glu Pro Pro Tyr180 185 190 Leu Asn Glu Thr Pro Leu Arg Ala Leu Tyr Leu Ile Ala Thr AsnGly 195 200 205 Thr Pro Lys Leu Lys Glu Pro Glu Asn Leu Ser Ser Ser LeuLys Lys 210 215 220 Phe Leu Asp Trp Cys Leu Cys Val Glu Pro Glu Asp ArgAla Ser Ala 225 230 235 240 Thr Glu Leu Leu His Asp Glu Tyr Ile Thr 245250 6 21 PRT human 6 Glu Ile Ser Ala Pro Ser Asn Phe Glu His Arg Val HisThr Gly Phe 1 5 10 15 Asp Gln His Glu Gln 20 7 21 PRT human; 7 Glu IleSer Pro Pro Ser Asp Phe Glu His Thr Ile His Val Gly Phe 1 5 10 15 AspAla Val Thr Gly 20 8 18 PRT yeast 8 Ile Ser Tyr Asn Ala Lys His Ile HisHis Val Gly Val Asp Ser Lys 1 5 10 15 Thr Gly 9 21 PRT human 9 Asp IleGly Ala Pro Ser Gly Phe Lys His Val Ser His Val Gly Trp 1 5 10 15 AspPro Gln Asn Gly 20 10 21 PRT yeast 10 Gly Val Ser Ser Pro Thr Asn PheThr His Lys Val His Val Gly Phe 1 5 10 15 Asp Pro Glu Thr Gly 20 11 8PRT human 11 Lys Lys Glu Leu Ile Ile Asn Glu 1 5 12 8 PRT human 12 ValGly Thr Pro Tyr Trp Met Ala 1 5 13 268 DNA mouse 13 aagcagcagcggcgcgagtt gctcttcaat gaggtggtga tcatgcggga ctaccggcac 60 gagaacgtggtggagatgta caacagctac ctggtgggtg acgaactctg ggtcgtcatg 120 gagttcctggaaggcggcgc cctcacggat attgtcaccc acaccaggat gaacgaggaa 180 cagatcgccgccgtgtgcct ggctgtgctt caggcgctgg ctgtgctcca cgcccagggt 240 gtcatccacagcgacataaa aacggaca 268 14 89 PRT mouse 14 Lys Gln Gln Arg Arg Glu LeuLeu Phe Asn Glu Val Val Ile Met Arg 1 5 10 15 Asp Tyr Arg His Glu AsnVal Val Glu Met Tyr Asn Ser Tyr Leu Val 20 25 30 Gly Asp Glu Leu Trp ValVal Met Glu Phe Leu Glu Gly Gly Ala Leu 35 40 45 Thr Asp Ile Val Thr HisThr Arg Met Asn Glu Glu Gln Ile Ala Ala 50 55 60 Val Cys Leu Ala Val LeuGln Ala Leu Ala Val Leu His Ala Gln Gly 65 70 75 80 Val Ile His Ser AspIle Lys Thr Asp 85 15 489 DNA mouse misc_feature (410)..(410) unkownnucleotide 15 acctggtggg tgacgaactc tgggtcgtca tggagttcct ggaaggcggcgccctcacgg 60 atattgtcac ccacaccagg taccataggg cagcctgctg gctcatgtgctccctggggt 120 ggaactggga ccctttaggc tctggtgata gacaagtgcc ctccagagtgtgggtggggc 180 agtgaggcca ggcacacagg atgggggtca tagcatcgtg gctccctgacccctgttgag 240 gcgggtcttt gtgacctctt gttgtctaaa gcagggtagg ggcctcttcactgcccactc 300 tcaccccagg gtgggatgcc caaggcagcg ctgagtgccc agttgctcctctgcccgcgc 360 aggatgaacg aggaacagat cgccgcccgt gtgcctggct tgtgcttcangcgctggctt 420 gtgctccacg cccagggtgt catccaccgt gacatcaaga gtgactctatcttgctgacc 480 catgatggc 489 16 6 PRT mouse 16 Gly Glu Gly Ser Thr Gly 15 17 6 PRT mouse 17 Ser Leu Val Gly Thr Pro 1 5

What is claimed is:
 1. An isolated mammalian PAK4 serine/threoninekinase comprising the amino acid sequence set forth in FIGS. 1A-1B (SEQID NO:2).
 2. An isolated mammalian PAK4 serine/threonine kinase whoseamino acid sequence consists of the amino acid sequence set forth inFIGS. 1A-1B (SEQ ID NO:2).